A solid phase synthesis process using a phosphoramidite method has been widely used for the chemical synthesis of nucleic acids such as DNA oligonucleotide, RNA oligonucleotide and the like. In the solid phase phosphoramidite method, nucleic acid synthesis is generally performed by the following steps.
First, a nucleoside to be the 3′-terminus of a nucleic acid to be synthesized is ester bonded to a cleavable linker such as succinyl group and the like via a 3′-OH group to be previously supported on a carrier for solid phase synthesis (nucleoside linker). Then, the carrier for solid phase synthesis, on which the nucleoside linker is supported, is placed in a reaction column and set on a nucleic acid automatic synthesizer.
According to the synthesis program of a nucleic acid automatic synthesizer, a synthesis reaction including the following steps is generally performed in a reaction column:    (1) a step of removing 5′-OH group of protected nucleoside with an acid such as trichloroacetic acid/dichloromethane solution and the like;    (2) a step of coupling nucleoside phosphoramidite (nucleic acid monomer) with a deprotected 5′-OH group in the presence of an activator (tetrazole etc.);    (3) a step of capping an unreacted 5′-OH group with acetic anhydride and the like; and    (4) a step of oxidizing phosphite with aqueous iodine and the like. This synthesis cycle is repeated, and an elongation reaction of oligonucleotide is performed from the 3′-terminus to the 5′-terminus direction, whereby a nucleic acid having the object sequence is synthesized.
Lastly, a cleavable linker is hydrolyzed with aqueous ammonia, methylamine solution and the like, and the synthesized nucleic acid is cleaved from the carrier for solid phase synthesis (non-patent document 1). In addition, cleavage with an aqueous sodium hydroxide solution containing NaCl has also been reported (non-patent document 2).
When the above-mentioned synthesis is performed, as mentioned above, nucleoside as a starting material to be the 3′-terminus needs to be supported on a carrier for solid phase synthesis in advance via a cleavable linker. The 3′-terminus varies depending on the sequence of a nucleic acid desired to be synthesized, 4 kinds of dA, dG, dC, dT are necessary in the case of DNA oligonucleotide, and 4 kinds of rA, rG, rC, rU are necessary in the case of RNA oligonucleotide. When a modified oligonucleotide is to be synthesized, the process becomes complicated since a carrier for solid phase synthesis previously made to support a modified nucleoside is necessary.
To overcome the aforementioned problems, a carrier supporting a universal linker for solid phase synthesis (universal support) has been proposed as a linker connecting the solid phase carrier and the starting material, instead of a nucleoside-succinyl linker and the like generally used heretofore. When a universal support is used, irrespective of the kind of the nucleoside or nucleotide to be the 3′-terminus of a nucleic acid desired to be synthesized, synthesis is started by reacting nucleoside phosphoramidide to be the 3′-terminus by the same steps as those of general nucleic acid automatic synthesis, and after synthesis, the object nucleic acid is cut out from the universal support by a method similar to a conventional method. Advantageously, as mentioned above, a carrier supporting various nucleoside-linkers for solid phase synthesis is not required.
Conventionally, as a method of cutting out RNA oligonucleotide from a universal support, a method including contact with alkylamine or aqueous ammonia/alkylamine mixed solution has been generally used (patent document 1). In recent years, moreover, a method including contact with ethanolamine has been developed (patent document 4). In these methods, however, RNA oligonucleotide cannot be cleaved well from the universal linker, a large amount of byproduct is produced, and the object RNA oligonucleotide cannot be cut out with good purity. That is, in a method of cutting out the object RNA oligonucleotide from a universal support, the solid phase carrier alone is cleaved, RNA oligonucleotide cannot be cleaved well from the universal linker, and a byproduct is sometimes produced. While the bond between the RNA oligonucleotide and the universal linker can be cleaved by a stronger nucleophilic reagent, more harsh temperature conditions and the like, the cleavage particularly promotes decomposition of RNA oligonucleotide, which is considered to decrease the yield of the object RNA oligonucleotide. On the other hand, when cutting out is performed under mild conditions in an attempt to increase the yield of RNA oligonucleotide, byproducts wherein RNA oligonucleotide and universal linker are not cleaved may result in large amounts. In addition, since the byproduct and the object RNA oligonucleotide have similar properties such as molecular weight, polarity and the like, purification thereof by separation and purification techniques such as chromatography and the like is problematically difficult. Mainly for these reasons, use of a universal support is difficult in the synthesis of RNA oligonucleotide.